TGF-ß2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

Lipidomic Phenotyping Of Human Small Intestinal Organoids Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

Current Practice in Bicistronic IRES Reporter Use: A Systematic Review.

Bicistronic reporter assays have been instrumental for transgene expression, understanding of internal ribosomal entry site (IRES) translation, and identification of novel cap-independent translational elements (CITE). We observed a large methodological variability in the use of bicistronic reporter assays and data presentation or normalization procedures. Therefore, we systematically searched the literature for bicistronic IRES reporter studies and analyzed methodological details, data visualization, and normalization procedures. Two hundred fifty-seven publications were identified using our search strategy (published 1994-2020). Experimental studies on eukaryotic adherent cell systems and the cell-free translation assay were included for further analysis. We evaluated the following methodological details for 176 full text articles: the bicistronic reporter design, the cell line or type, transfection methods, and time point of analyses post-transfection. For the cell-free translation assay, we focused on methods of in vitro transcription, type of translation lysate, and incubation times and assay temperature. Data can be presented in multiple ways: raw data from individual cistrons, a ratio of the two, or fold changes thereof. In addition, many different control experiments have been suggested when studying IRES-mediated translation. In addition, many different normalization and control experiments have been suggested when studying IRES-mediated translation. Therefore, we also categorized and summarized their use. Our unbiased analyses provide a representative overview of bicistronic IRES reporter use. We identified parameters that were reported inconsistently or incompletely, which could hamper data reproduction and interpretation. On the basis of our analyses, we encourage adhering to a number of practices that should improve transparency of bicistronic reporter data presentation and improve methodological descriptions to facilitate data replication.